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H. Janovjak
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P3.03 - Poster Session with Presenters Present (ID 473)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 2
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.03-002 - Inducible Changes in Cell Morphology and Gene Expression Reflecting the Histological Subtypes of Mesothelioma (ID 5405)
14:30 - 14:30 | Author(s): H. Janovjak
- Abstract
Background:
Malignant pleural mesothelioma (MPM) represents an aggressive malignancy with dismal prognosis and limited therapeutic options. MPM occurs in three main histological subtypes: epithelioid, sarcomatoid and biphasic, which are characterized by differences in morphological growth pattern, aggressiveness and patient prognosis. However, the mechanisms and causes responsible for the different cell morphologies are poorly understood. Epithelial-mesenchymal transition (EMT) has been implicated in cancer progression and chemoresistance, but its role in MPM is not well understood. Fibroblast growth factor (FGF) signals promote cell growth, survival and aggressiveness in several tumors including mesothelioma. Aim of this study was to characterize growth factor-induced, EMT-like changes with respect to the MPM histological subtypes.
Methods:
Morphological and behavioral changes of treated cell models were analyzed by morphometry, immunoblotting and functional assays. Alterations in gene or microRNA expression were evaluated via qPCR and array hybridization. Pathway enrichment analysis was based on KEGG.
Results:
In several cell lines established from biphasic MPM, treatment with FGF2 and EGF induced morphological changes reminiscent of EMT and aggressive behavior such as scattering, increased migration, proliferation and invasiveness. Inhibition of the fibroblast growth factor receptors (FGFR) or the MAPK axis via small-molecule inhibitors could prevent these changes and, in cell lines with sarcomatoid-like shape, reverse scattering and induce a more epithelioid morphology. Comparable results were obtained using an engineered FGFR1 enabling contactless activation via blue light. Analyses of genes and microRNAs regulated by FGF2 or EGF showed an overlap with previously established EMT markers but also identified several novel potential markers such as MMP1, ESM1, ETV4, PDL1, ITGA6 or BDKRB2. Blocking the FGFR or MAPK pathways resulted in the opposite regulation of these genes. Inhibition of MMP1 via siRNAs or pharmacological inhibitors prevented FGF2-induced scattering and invasiveness. In unsupervised clustering, the gene expression profiles of solvent- or cytokine-treated cells were associated with those of epithelioid and sarcomatoid MPM, respectively. Immunohistochemistry showed an association of MMP1 as well as phospho-ERK with the sarcomatoid part of tissue specimens from biphasic tumors. Pathway enrichment analysis of differentially expressed genes as well as the targets of altered microRNAs after FGF2 treatment showed that the regulated genes are assigned to categories important for cell growth and aggressive behavior.
Conclusion:
Our data characterize FGFR-mediated signals as important players in MPM aggressiveness and the morphological and behavioral plasticity of mesothelioma cells, leading to a better understanding of the link between the MPM histological subtypes and their influence on patient outcome.
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P3.03-006 - Optical Control of Growth Factor Receptors to Advance Signal Transduction Research and Drug Screening (ID 5358)
14:30 - 14:30 | Author(s): H. Janovjak
- Abstract
Background:
Growth factor receptors are central elements of signal transduction pathways and increasingly important targets for anticancer drugs. In recent years naturally occurring light sensitive protein domains (LSPDs) from different kingdoms of life have been used to generate genetically encoded chimeric signalling molecules that can be activated reversibly and with spatiotemporal precision by light. The development of such optogenetic tools has led to a plethora of new discoveries in the neurosciences but has received comparably little attention in cancer research - partly due to a lack of appropriate tools. Our aim was therefore to generate synthetic growth factor receptors that can be activated with light and allow fine-tuned control of growth factor-associated signal transduction pathways.
Methods:
To generate receptor tyrosine kinases (RTKs) that can be optically activated (Opto-RTKs), intracellular domains of RTKs were fused to LSPDs of the light-oxygen voltage (LOV) family from various species. The resulting chimeric receptors were tested for light-dependent activation of signal transduction by reporter gene assays, immunoblotting and various cell biological tests assessing DNA synthesis, epithelial mesenchymal transition (EMT) and angiogenesis.
Results:
Three of the tested LOV domains enabled light-dependent receptor dimerisation and activation of the corresponding signal transduction pathways when fused to the intracellular domains of FGFR1, EGFR, RET, c-Met or ROS1. Opto-RTKs enabled stringent control of the MAPK, PI3K and PLCĪ³ pathways. Signal activation could be spatially confined to illuminated regions of culture plates and signals rapidly subsided after cessation of illumination. Light was able to replace FGF2 for the induction of cell proliferation and EMT in mesothelioma cells and VEGF for the stimulation of angiogenic sprouting in endothelial cells. Moreover, Opto-RTKs enabled light-assisted screening for small molecule inhibitors of EGFR, FGFR1 and the orphan RTK ROS1.
Conclusion:
Our optogenetic approach allows light-mediated control of growth factor receptors representing clinically relevant drug targets. Opto-RTKs enable dissection of dynamic signals with increased spatiotemporal resolution and open new possibilities for drug screening. Transfer of the design principle to additional membrane receptors is ongoing.
